Vaccinia virus is an orthopoxvirus, which is a class of large, DNA-containing animal viruses that replicate primarily in the cytoplasm of infected cells. It has about 200 genes, which are expressed in a complex, regulated, developmental program. One of these genes codes for a viral thymidine kinase (tk), which is expressed early in infection and then repressed. The object of this proposal is to study the location, structure, mechanisms of control, and function of the vaccinia virus tk gene, in order to understand better the basic principles involved in the control of gene expression. Messenger RNA extracted from vaccinia virus-infected cells can be translated in vitro to yield enzymically active thymidine kinase. We will exploit this capability to determine the activity, size, and structure of the tk mRNA, both in infected cells and in RNA synthesized by viral cores in vitro. The physical location of the tk gene within the viral genome will be mapped by translation of mRNA selected by hybridization to defined fragments of viral DNA. DNA-mediated marker rescue of mutants deficient in tk will be used as an alternative mapping procedure. Recombinant plasmids containing the tk gene will be identified or constructed, and used to confirm and refine the map position of the gene. Sub-cloning of the gene in bacteriophage M-13 DNA will be performed, to provide a single-stranded DNA for use in sequence determination and as a hybridization probe to assay the synthesis, accumulation, and degradation of tk transcripts in infected cells. Promoter negative mutants of the virus will be sought and characterized, and the mechanisms that control the repression of thymidine kinase expression half way through infection will be elucidated. The purified viral gene will be used to study the requirements for the successful biochemical transformation of animal cells with a gene that does not normally occupy the nuclei of infected cells.